Abstract
BackgroundThough massively parallel sequencing has been widely applied to noninvasive prenatal screen for common trisomy, the clinical use of massively parallel sequencing to noninvasive prenatal diagnose monogenic disorders is limited. This study was to develop a method for directly determining paternal haplotypes for noninvasive prenatal diagnosis of monogenic disorders without requiring proband’s samples.MethodsThe study recruited 40 families at high risk for autosomal recessive diseases. The targeted linked-read sequencing was performed on high molecular weight (HMW) DNA of parents using customized probes designed to capture targeted genes and single-nucleotide polymorphisms (SNPs) distributed within 1Mb flanking region of targeted genes. Plasma DNA from pregnant mothers also underwent targeted sequencing using the same probes to determine fetal haplotypes according to parental haplotypes. The results were further confirmed by invasive prenatal diagnosis.ResultsSeventy-eight parental haplotypes of targeted gene were successfully determined by targeted linked-read sequencing. The predicted fetal inheritance of variant was correctly deduced in 38 families in which the variants had been confirmed by invasive prenatal diagnosis. Two families were determined to be no-call.ConclusionsTargeted linked-read sequencing method demonstrated to be an effective means to phase personal haplotype for noninvasive prenatal diagnosis of monogenic disorders.
Highlights
Though massively parallel sequencing has been widely applied to noninvasive prenatal screen for common trisomy, the clinical use of massively parallel sequencing to noninvasive prenatal diagnose monogenic disorders is limited
Our group has employed a proband-based method for resolving parental haplotypes and successfully applied this method to noninvasive prenatal diagnosis (NIPD) of Duchenne muscular dystrophy (DMD) [7], congenital adrenal hyperplasia (CAH) [8], maple syrup urine disease (MSUD) [9], hyperphenylalaninemia [10] and spinal muscular atrophy (SMA) [11]
We demonstrated direct haplotyping of NIPD based on linked‐read sequencing is accurate for the prediction of fetal pathogenic variants of DMD [20]
Summary
Though massively parallel sequencing has been widely applied to noninvasive prenatal screen for common trisomy, the clinical use of massively parallel sequencing to noninvasive prenatal diagnose monogenic disorders is limited. Serval studies have reported specific haplotype building methods such as clone pool dilution sequencing [12], contiguity-preserving transposition sequencing [13], targeted locus amplification (TLA) [14], HaploSeq [15] and long fragment read (LFR) technology [16] These approaches need complex experimental operations and are time consuming and associated with a low success rate. In order to further improve the success rate and accuracy of haplotype phasing, microfluidics-based linked-read sequencing technology and TLA-based phasing were utilized to phase parental DNA directly [18, 19] The former approach combined the whole-genome sequencing (WGS) and linked-read sequencing method and succeeded in predicting fetal inherited variants in 12 of 13 pregnancies. We speculated that the linked-read sequencing combined with targeted sequencing using the above probes would expand the list of single gene disorders and reduce the cost compared with the wholegenome sequencing
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